Historically the discovery and optimization of candidate compounds for development as drugs has been extraordinarily expensive and time-consuming. Although the relatively new approach of "rational drug design" has promise for the future, the pharmaceutical industry has generally relied on mass screening of many-membered "libraries" of chemical compounds for the identification of "lead" compounds worthy of further study and structure-activity relationship (SAR) work. To meet this need high-throughput screening (HTS) technology has been developed that permits pharmaceutical companies to evaluate hundreds of thousands of individual chemical entities per year. Typically, these screens involve measuring some interaction (e.g., binding) between a biological target such as an enzyme or receptor and chemical compounds under test. The screens generally commence with the addition of individual compounds (or mixtures of compounds) to the individual wells in a 96 or higher-well "microtiter" plate that contains the biological target of interest (e.g., a receptor, enzyme or other protein). Ligand/receptor binding or other interaction events are then deduced by, for instance, various spectrophotometric techniques. Those chemical entities that exhibit promise in initial screens (e.g., that bind a biological target with some threshhold affinity) are then subjected to chemical optimization, SAR work, other types of testing, and, if warranted, eventual development as drugs.
Now that HTS has simplified and made more cost-effective the task of determining whether large chemical libraries contain promising lead compounds or "hits", many pharmaceutical companies are limited not by their ability to screen candidate compounds but rather by their ability to synthesize them in the first place. At one point, most pharmaceutical companies relied on their historical collections of natural products and individually synthesized chemical entities as compound libraries to be subjected to mass screening. However, expanding these libraries--especially with a view toward increasing the "diversity" of the chemical space that they probe--has proven problematic. For instance, the cost of having a synthetic organic or medicinal chemist synthesize individual molecules in a serial fashion has been estimated to be several thousand dollars, and this is obviously a painstakingly slow process.
Thus, the advent of high-throughput screening has created a need for correspondingly high-throughput chemical synthesis (HTCS) to feed this activity. "Combinatorial chemistry" and related techniques for high-throughput parallel syntheses of large chemical libraries were created in response to this need (Gallop, M. A. et al, "Applications of Combinatorial Technologies to Drug Discovery: 1. Background and Peptide Combinatorial Libraries," J. Med. Chem., 37 (9) :1233-1251 (1994); Gordon, E. M. et al, "Applications of Combinatorial Technologies to Drug Discovery: 2. Combinatorial Organic Synthesis, Library Screening Strategies, and Future Directions," J. Med. Chem., 37 (10):1385-1401 (1994); Baum, R. M., "Combinatorial Approaches Provide Fresh Leads for Medicinal Chemistry," C&E News, pp. 20-26, Feb. 7, 1994; Plunkett, M. J. et al, "Combinatorial Chemistry and New Drugs," Scientific American, 276 (4):68-73 (1997); Borman, S., "Combinatorial Chemistry," C&E News, pp. 44-67, Apr. 6, 1998). To simplify the separation of intermediate compounds during multistep organic syntheses, much of this chemistry is generally performed while the compound being synthesized is covalently immobilized on small support beads. Once the chemical building blocks have been properly assembled, the desired compounds are usually cleaved from their supports (often highly swellable polymeric resins) before being carried through to HTS.
Various definitions of "combinatorial chemistry" and "combinatorial synthesis" have been proposed and are in current use. Some synthesis strategies (e.g., "split-and-mix") are truly "combinatorial" in nature and have as their hallmark the ability to produce very large libraries; indeed, as many as a million library members can be synthesized in a modest number of reactions (and correspondingly small number of reaction vessels) by virtue of the exponential mathematics involved. One of the several limitations of such approaches, however, is the difficulty of identifying the particular individual chemical species responsible for any activity measured in an assay of what is generally a mixture of compounds.
Other approaches such as high-throughput parallel synthesis are typically used to produce somewhat smaller chemical libraries containing, for example, from several hundred to several hundred thousand individual compounds. Here, discrete compounds (and occasionally mixtures) are spatially segregated during chemical synthesis so no ambiguity exists as to the identity of any compound producing a "hit." However, parallel synthesis requires that chemical reactions be conducted in parallel in a relatively large number of reaction vessels, thus placing a premium on the ability to automate and improve the speed and efficiency of the synthetic process.
The terms "combinatorial chemistry," "combinatorial synthesis," and "parallel synthesis" are used herein synonymously and interchangeably to denote various high-throughput approaches for the preparation of chemical libraries, whether by solid-phase or solution-phase synthesis. Although the present invention is described primarily in terms of its capabilities for solid-phase synthesis, the invention is not so limited. Similarly, the present description focuses principally on the parallel synthesis of discrete compounds (i.e., one chemical entity per reaction chamber or vessel), although truly combinatorial, split-and-mix synthesis as well as the synthesis of compound mixtures can be performed equally well with the apparatus and method of the present invention.
There currently exist several different approaches for the parallel, solid-phase synthesis of discrete compounds, with somewhat different types of apparatus being best suited to each approach. The approaches described here can be referred to as "spatially addressable" strategies for the reason that, generally, each unique compound is synthesized (and addressable) at a separate point in space--that is, one compound is synthesized per reaction vessel in a multi-vessel "reaction block". The devices and equipment used to execute these different spatially addressable synthesis strategies differ considerably in terms of their degree of sophistocation, automation, and cost--ranging from fully automated robotic synthesizers presently costing as much as several hundred thousand dollars to simple, disposable, inexpensive 96-well microtiter plates modified for chemical synthesis.
Most high-throughput chemical syntheses (HTCS) performed in the context of combinatorial chemistry and parallel synthesis are presently conducted in multi-vessel reaction assemblies often referred to as "reaction blocks" by virtue of their monolithic construction. In most solid-phase syntheses, the compound being constructed is covalently attached to resin beads and so many of these multi-vessel reaction blocks include provision for a porous frit to retain the polymer resin beads (and compounds attached thereto) in the reaction vessel during the multiple resin washing steps that are used to remove excess reagents (e.g., building blocks, solvents, catalysts, etc.) after individual reaction steps. In some designs the compounds being synthesized are immobilized on porous/solvent-swollen "pins" that extend into individual reaction chambers (Geysen, H M et al, Proc.Natl.Acad.Sci.USA, 81:3998 (1984); U.S. Pat. No. 4,708,871 (1987)). Other approaches utilize small porous sacks or "teabags" to contain the resin (Houghten, R A, Proc. Natl.Acad.Sci.USA, 82:5131 (1985)).
Since most chemical libraries synthesized by HTCS are destined for mass screening, and since the 96-well plate is the standard platform for assay of biological activity by HTS, most reaction vessel asssemblies or reaction blocks for combinatorial synthesis contain either 96 vessels or a simple fraction of that number (e.g. 48 or 24). One of the first automated instruments specifically designed and marketed with combinatorial chemistry in mind is the Model 396 MPS ("multiple peptide synthesizer", a name reflective of the original market for the instrument) manufactured by Advanced ChemTech, Inc. (Louisville, Ky.). Subsequent to the introduction of the Model 396, the Advanced ChemTech product line was expanded to include other instruments, e.g. the Model 496. In both of the instruments, syntheses are conducted in a plastic (Teflon.RTM.) reaction block in which 96 discrete reaction chambers or vessels have been machined. One or more of these reaction blocks is placed within the working space of an automated liquid handling system or "robot" capable of delivering various solvents and reagents to discrete reaction vessels. A frit at the base of each reaction vessel retains resin (and compound) during resin washing steps, with fluids being removed from the reaction chambers through a siphon arrangement. The siphon system inherently limits usefulness of the device in terms of the pressures at which it can successfully be operated--and, in particular, the maximum pressure differences that can be tolerated before the contents of the reaction vessels leak out.
Since the Advanced ChemTech automated synthesizers and reaction blocks were among the first to market, they have been used extensively in combinatorial chemistry laboratories. However, the 96-vessel reaction blocks have a number of drawbacks. Due to their monolithic, machined construction, they are expensive to manufacture and damage to any part of a block can cause the entire block to be unusable. Moreover, because they are machined from plastic materials (to provide inert and solvent-resistant vessel surfaces in contact with the chemistries being conducted within them), the blocks have poor thermal conductivity. Thus heating or cooling to reaction conditions can be slow. Another limitation of these reaction blocks is a difficulty of achieving gas and vapor-tight seals, especially where aggressive and/or volatile solvents and elevated reaction temperatures are utilized.
Finally, the Model 396 and 496 reaction blocks are physically large and heavy. Their size and weight interfere with their placement on and removal from the platforms of the robotic workstations used to address them. It is often desirable to be able to move the reaction blocks, e.g. to permit reactions to be conducted off-line thereby freeing up expensive workstation space, but the cumbersome nature of the Model 396 and 496 reaction blocks makes this inconvenient. Just as significant a drawback, however, is that, despite containing 96 reaction vessels (and compounds or mixtures thereof), it is impossible to cleave these compounds directly into the wells of the much smaller (33/8".times.5") 96-well microtiter plates most commonly used for compound storage and assay. The footprint of the Model 396 and 496 reaction blocks is incompatible with the footprint of conventional microtiter plates, which makes it impossible to address these reaction blocks with other automated equipment which has been designed around the standardized platform of the 96-well plate. Also, there is no convenient way of "reformatting" or transferring compounds to such microplates if they are initially cleaved into test tubes or vials.
Advanced ChemTech manufactures still other reaction blocks and automated synthesizers including a Model 440 system based on a reaction block containing 40 8-mL reaction vessels, as well as lower-end semi-automated and manual systems marketed as their ReacTech and LabTech product lines. These related products suffer from many of the same disadvantages and limitations discussed above.
Another reaction block design is described in U.S. Pat. No. 5,609,826, of Ontogen Corp. Ontogen's "OntoBLOCK" reaction block system is comprised of two similar "alpha" and "beta" blocks, each of which holds 48 2-mL reaction chambers; a pair of two blocks can deliver cleaved compounds directly into 96-well plates. Individual reaction chambers, each fitted with porous bottom frits, are polymeric and removable; in use, they slip into an anodized aluminum block that serves to hold the reaction chambers in place and have S-shaped trap tubes for draining vessel contents. The reaction blocks can be closed with elastomeric seals to maintain an inert atmosphere and/or to contain volatile solvents at elevated reaction temperatures. The Ontogen reaction blocks are fitted with metal pins to facilitate securing the blocks on complementary docking stations. The pins also permit the blocks to be moved and addressed by robotic handling equipment. The S-trap tube inherently limits usefulness of the device in terms of the maximum pressure difference that can be tolerated before the contents of the reaction vessels leak out.
A somewhat different type of highly automated instrument for HTCS--again, with origins in solid-phase peptide synthesis--is exemplified by the Nautilus.TM. 2400 organic synthesizer manufactured by Argonaut Technologies, Inc. (San Carlos, Calif.). This instrument is not based on a reaction block per se. Rather it directs reagents through an assembly of valves to 24 individual glass reaction vessels mounted on the synthesizer. Like the Advanced ChemTech synthesizer, the Nautilus 2400 instrument is expensive and although capable of performing a wide range of chemistries is limited in terms of the number of syntheses that can be conducted simultaneously. Argonaut's Quest.TM. 210 manual synthesizer is similar in concept and is designed to perform but 20 reactions in parallel.
Several other reaction block designs and automated systems based upon them combine some of the above-cited features and are regarded as "hybrids" in certain respects. For example, (i) the CombiTec synthesizer developed by Tecan (Research Triangle Park, N.C.) and marketed by Perkin-Elmer (Foster City, Calif.), (ii) the Pathogenesis/RAM.TM. synthesizer developed and marketed by Bohdan Automation, Inc. (Mundelein, Ill.), and (iii) the CombiSyn.TM. synthesizer under development by CombiChem, Inc. (San Diego, Calif.) all rely on glass reaction vessels fitted to and/or within a manifold with provisions for fluid supply and withdrawal. Although addressable by liquid handling robots, the CombiSyn.TM. synthesizer relies on an arrangement of fluid valves (as in Argonaut's Nautilus.TM. product) to supply and remove reagents and solvents from the reaction vessels. All rely on porous frits to retain resin within individual reaction vessels.
At the other extreme from these expensive fully automated synthesizers there exists several simpler and less expensive products marketed for combinatorial synthesis that are based largely on modifications to the standard 96-well microtiter plate. For example, Polyfiltronics/Whatman (Rockland, Mass.) and Millipore (Bedford, Mass.) both market solvent-resistant 96-well plates fitted with solvent-resistant filters for resin/solution retention. Although synthetic versatility is limited--they cannot readily be sealed--they are inexpensive to manufacture and thus are disposable. However, they are unsuited for performing multistep chemical reactions, especially at elevated temperature and/or with volatile solvents.
Other "low-end" product offerings include the MultiReactor.TM. available from RoboSynthon, Inc. (Burlingame, Calif.) and the Multiblock available from CSPS (Tucson, Ariz.). However, the former product is limited to solution-phase chemistries, while the latter is based on the use of an unwieldy array of plastic syringes pressed into service as chemical reactors. The SPS (Solid Phase Synthesis) Reactor offered by J-Kem Scientific, Inc. (St. Louis, Mo.) is similar in that it relies upon an array of syringe barrels fitted with porous plastic frits for resin retention.
Several other reaction blocks designed specifically for combinatorial/parallel synthesis fall between the extremes represented by "high-end", fully automated organic synthesizers on the one hand and "low-end" reaction assemblies based on plastic microtiter plates and syringe arrays on the other hand. Typical of these products are the FlexChem.TM. reaction block system developed by Robbins Scientific Corp. (Sunnyvale, Calif.) and the Calypso System.TM. offered by Charybdis Technologies, Inc. (Carlsbad, Calif.).
The Robbins Scientific FlexChem.TM. synthesis/filtration block has a single molded polypropylene unit that contains 96 2-mL reaction wells with porous polyethylene frits (for resin retention) pressed into the bottom of each chamber. The one-piece plastic block can be sealed top and bottom against elastomeric septa or gaskets by clamping it tightly between two metal sealing covers--the top one of which is provided with beveled holes to permit access to individual reaction wells via a septum-piercing needle of a robotic liquid handler. The synthesis/filtration block has the same footprint as a standard 96-well microtiter plate so that cleaved compounds can be transferred directly to same. A vacuum manifold fits the bottom of the reaction block and permits withdrawal of excess reagents and reaction and wash solvents from the reaction wells inbetween reaction steps. A second, larger vacuum manifold permits recovery of cleaved compounds into 96-well plates housed therein. The Robbins Scientific reaction block can also be used in conjunction with a rotating incubator (for resin agitation and heating during reaction steps) and a 96-channel dispenser (for addition of wash solvent after reaction steps).
A major disadvantage of Robbins Scientifics' FlexChem.TM. synthesis/filtration block is that it requires considerable manual intervention on the part of the combinatorial chemist--especially in the time-consuming and laborious steps of resin washing. Thus, operations can at best be semi-automated. Additionally, the FlexChe.TM. reactor blocks are constructed from a polypropylene material which has poor thermal conductivity and reportedly contains high levels of extractables. Also, it is difficult to seal the plastic block tightly and reliably against its top and bottom metal cover plates--the eight spring clamps and collar arrangement are unwieldy and inconvenient to use. Finally, the molded plastic reaction blocks--while much less expensive than some--are still expensive enough to invite reuse and so are not truly disposable. Over time, especially with repeated exposure to solvents, the polypropylene blocks tend either to become brittle and break--or to soften and lose mechanical rigidity.
The Calypso System.TM. of Charybdis Technologies, Inc., is based on reusable, machined PTFE Teflon.RTM. blocks that contain 96 2-mL reaction vessels or "wells" spaced as per the standard microtiter plate format. (Other higher-capacity blocks are available that contain either 48 5-mL wells or 24 10-mL wells.) Separate reaction blocks are required for solid-phase and solution-phase syntheses. The reaction blocks come either with bottom filtration means for solid-phase syntheses or with closed bottoms for solution-phase chemistries. Like the Robbins Scientific product, Charybdis' block is sealed top and bottom against rubber septa (to internal pressures as high as 30 psi) by clamping it between metal plates with the aid of bolts. Again, the top metal plate is perforated to permit accessing individual reaction chambers via the needle of a robotic liquid handling system. Provisions for inerting the block (e.g., with nitrogen) are also made.
As with Robbins Scientifics' FlexChem.TM. synthesis/filtration block, a major drawback of the Charybdis Calypso System.TM. is the need for operator intervention during time-consuming resin washing operations; i.e., removal of wash solvents from the block is a manual operation. Also, the Calypso System.TM., while less expensive than the fully automated synthesizers reviewed above, is significantly more expensive than many of the "low-end" reaction blocks based on modified 96-well microtiter plates.
Accordingly, there exists a need in the art for a modestly priced yet versatile reaction block with which to conduct combinatorial chemistry and high-throughput parallel syntheses. Ideally, the reaction block should present only chemically inert surfaces to the reactant solutions so that compounds submitted for HTS are free of contaminants and extractables; the materials of which the block is constructed must resist aggressive solvents and severe reaction conditions (e.g., elevated temperatures); and the block should be constructed of a high-thermal-conductivity material to facilitate rapid and uniform heat transfer. Individual reaction vessels in the reaction block need to have fluid capacity of about 2 mL or greater and preferably can be fitted with porous plastic frits for resin retention. Means for retaining or removing the liquid contents of individual reaction vessels should be positive, reliable, and convenient. The reaction block must also be purgable with, e.g., inert gases.
There also remains a need in the art for reaction blocks and compound transfer tools with footprints corresponding to that of the standard 96-well microtiter plate. Moreover, the design of these components should facilitate transfer or reformatting of the contents of individual reaction vessels to the corresponding wells in the microtiter plates. Those prior-art reaction blocks which do feature the size and layout of microtiter plates suffer from one or more other serious drawbacks that prevent them from being regarded as appropriate solutions to the above-mentioned unsolved problems.
Finally, there remains a need in the art for relatively low-cost reaction blocks, wash plates, and associated wash stations and other workstations that minimize requirements for operator intervention during the most time-consuming steps in combinatorial/parallel synthesis, especially those steps associated with resin washing.
The present invention fulfills these and other heretofore unmet needs and provides cost-effective productivity tools for use in the construction of compound libraries useful in drug discovery.